Detecting Borrelia in Tick, Rodent, and Human Samples

Genomic DNA extracted from pooled ticks, rodent tissue, and patient’s whole blood were screened for the presence of Borrelia using a genus-based TaqMan real-time PCR assay targeting the Borrelia 16S rRNA gene [28 (link)]. Borrelia-positive samples were further characterized by sequencing the flagellin (flaB) and 16S rRNA genes. The primer and probe sequences and conditions used in this study has been described previously by Takhampunya et al. [18 (link)]. The PCR products were purified using ExoSap-IT PCR Product Cleanup Reagent (Applied Biosystems, Foster City, CA) and sequenced using a SeqStudio genetic analyzer (Applied Biosystems). Raw sequences were edited and assembled using DNA Sequencher ver. 5.1 (Gene Code Corporation, Ann Arbor, MI).

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Takhampunya R., Longkunan A., Somchaimongkol S., Youngdech N., Chanarat N., Sakolvaree J., Tippayachai B., Promsathaporn S., Phanpheuch B., Poole-Smith B.K., McCardle P.W, & Lindroth E.J. (2023). Borrelia miyamotoi a neglected tick-borne relapsing fever spirochete in Thailand. PLOS Neglected Tropical Diseases, 17(2), e0011159.

Publication 2023

ExoSap-IT PCR Product Cleanup Reagent

16s rrna Assay Blood Borrelia Flagellin Gene code Genes Genomic Patient Primer Real time pcr Rodent Rrna genes Seqstudio Ticks Tissue

Corresponding Organization : United States Army

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Variable analysis

independent variables

Genomic DNA extracted from pooled ticks
Genomic DNA extracted from rodent tissue
Genomic DNA extracted from patient's whole blood

dependent variables

Presence of Borrelia using a genus-based TaqMan real-time PCR assay targeting the Borrelia 16S rRNA gene
Characterization of Borrelia-positive samples by sequencing the flagellin (flaB) and 16S rRNA genes

control variables

Primer and probe sequences and conditions used in this study as described previously by Takhampunya et al. [18 (link)]
PCR product purification using ExoSap-IT PCR Product Cleanup Reagent (Applied Biosystems, Foster City, CA)
Sequencing using a SeqStudio genetic analyzer (Applied Biosystems)
Raw sequence editing and assembly using DNA Sequencher ver. 5.1 (Gene Code Corporation, Ann Arbor, MI)

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

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